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4DRINKING WATERDISTRIBUTION SYSTEMS:BIOFILM MICROBIOLOGY
4.1 INTRODUCTION
Following water treatment by processes mentioned in Chapters 2 and 3, water is dis-
tributed through a network of pipes to reach customers. The water distribution system
(WDS), an essential component of drinking water treatment, is the “workhorse” that
carries drinking water from the plant to the customers. The daily production in the
United States is estimated 34 billion gallons of drinking water that flows through 1.8
million miles of distribution pipes. It is estimated that approximately two-third of
the pipes are made of plastic, mostly polyvinyl chloride (PVC). Many waterborne
outbreaks are attributed to the degradation of water quality in noncovered water reser-
voirs and in WDSs through cross-connections, main breaks (an estimated 240,000
water distribution main breaks per year in the United States; Lafrance, 2011), con-
tamination during construction or repair, back siphonage (National Research Council,
2006), or negative pressure events (Gullick et al., 2004; NRC, 2006).
Drinking water regulations mostly concerns water quality at the end of the water
treatment plant but the water quality can deteriorate during storage and transport
through water distribution pipes and in indoor plumbing before reaching the con-
sumer. More recently, US federal regulations addressed water quality in distribution
systems through rules covered by the Safe Drinking Water Act (SDWA).
In the long history of water distribution which goes back to 2000 BC, pipes
were made of a wide range of materials which include aqueducts, terracotta, wood,
copper, lead, bronze, bamboo, and stone. Nowadays, piping materials include cast
iron, ductile iron, stainless steel, concrete, asbestos-cement, or PVC, polyethylene,
and medium density polyethylene (MDPE) (Bachmann and Edyvean, 2006; Berry
et al., 2006; Lafrance, 2011; Walski, 2006; Percival et al., 2000). However, some of
Microbiology of Drinking Water Production and Distribution, First Edition. Gabriel Bitton.© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.
91
92 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
the piping materials lead to corrosion problems (e.g., cast iron and copper pipes) or
to leaching of toxic materials (e.g., PVC pipes).
Some major problems concerning WDSs are the following:
� Microbial growth� Pathogen survival and growth� Nitrification problems� Biocorrosion
Deterioration of water distributions systems due to leakage from cracks and joints
or back siphonage may lead to drinking water contamination and disease outbreaks.
In the United States, approximately 18% of the outbreaks reported for public water
systems are due to contamination of the WDS by microbial pathogens and toxic
chemicals (Craun, 2001). The contribution of WDSs to acute gastrointestinal illness
(AGI) incidence was demonstrated by epidemiological studies which showed that the
incidence was higher among consumers who drank from home taps than those who
drank treated water bottled at the water treatment plant (Payment et al., 1991a, 1997).
This shows that WDS and in-premise plumbing may contribute to disease in the
community. The risk of viral AGI from nondisinfected groundwater-based drinking
WDSs exceeded the U.S. EPA acceptable risk of 10−4 episodes/person-year. An
extrapolation of the results to the United States as a whole shows an AGI risk of
470,000 to 1,100,000 episodes/year nationwide (Lambertini et al., 2012; U.S. EPA,
1989).
Drinking water deterioration inWDS has several causes (LeChevallier et al., 2011;
Smith, 2002; Sobsey and Olson, 1983):
� Improperly built and operated storage reservoirs which should be covered to
prevent airborne contamination and to exclude animals.� Loss of disinfectant residual.� Back siphonage and cross-contamination.
Some of the consequences are
� Public health problems due to the excessive growth and colonization ofwater dis-
tribution pipes by bacteria and other organisms, some of which are pathogenic.
Microbial growth depends on the availability of nutrients (see Chapter 6)� Microbial regrowth in storage reservoirs� Protection of pathogens from disinfectant action� Taste and odor problems due to the growth of algae, actinomycetes and fungi
(see Chapter 5)� Corrosion problems
Drinkingwatermay contain humic and fulvic acids, aswell as easily biodegradable
natural organics such as carbohydrates, proteins, and lipids. The presence of dissolved
organic compounds in finished drinking water is responsible for several problems,
BIOFILM DEVELOPMENT IN WDSs 93
among which are taste and odors, enhanced chlorine demand, trihalomethane forma-
tion, and bacterial colonization of water distribution lines (see Chapter 6 for more
details). Water utilities should maintain the distribution system by periodically flush-
ing the lines to remove sediments, excessive bacterial growth, and encrustations due
to corrosion.
4.2 BIOFILM DEVELOPMENT IN WDSs
4.2.1 Introduction
Biofilms develop at solid-water interfaces and are widespread in natural environments
as well as in engineered systems. They are ubiquitous and are commonly found in
trickling filters, rotating biological contactors, activated carbon beds, distribution pipe
surfaces, groundwater aquifers, aquatic weeds, tooth surfaces (i.e., dental plaque),
dialysis units, dental unit waterlines, and indwelling medical devices (IMDs) such
as catheters, pacemakers, orthopedic implants, endotracheal tubes, prosthetic joints,
and heart valves (Anwar and Costerton, 1992; Characklis, 1988; Hamilton, 1987;
Schachter, 2003; Thomas et al., 2004; Walker and Marsh, 2007; van der Wende
and Characklis, 1990). For example, in the United States, the risk of infection from
bladder catheters ranges from 10% to 30% while the risk from cardiac pacemakers
is 1–5% (Thomas et al., 2004).
Biofilms are relatively thin layers (up to a few hundred microns thick) of attached
microorganisms that form microbial aggregates and grow on surfaces. Biofilms
formed by prokaryotic microorganisms resemble in some ways tissues formed by
eukaryotic cells (Costerton et al., 1995; Wingender and Flemming, 2011). The multi-
cellular behavior is due to the fact that cells within the biofilm interact and coordinate
their activities via direct cell-to-cell contact or by releasing signaling molecules
(see quorum sensing in Section 4.2.5) (Branda and Kolter, 2004). Observations of
living biofilms by scanning confocal laser microscopy have helped in our understand-
ing of biofilm structure. Biofilm microorganisms form microcolonies separated by
open water channels (Stoodley et al., 2002; Donlan, 2002). Liquid flowing through
these channels allow the diffusion of nutrients, oxygen, and antimicrobial agents
into the cells (Donlan, 2002). Some bacteria, such as Staphylococcus epidermidis,form honeycomb-like structures (Schaudinn et al., 2007) that provide mechanical
stability to the biofilm, help lower the energy costs of individual cells and maximize
nutrient absorption. Biofilms also include corrosion by-products, organic detritus,
and inorganic particles such as silt and clay minerals. They contain heterogeneous
assemblages of microorganisms (Figure 4.1), depending on the chemical composition
of the surface, the chemistry of finished water, and oxido-reduction potential in the
biofilm. They take days to weeks to develop, depending on nutrient availability and
environmental conditions. Biofilm growth proceeds up to a critical thickness (approx-
imately 100–200 μm) when nutrient diffusion across the biofilm becomes limiting.
The decreased diffusion of oxygen is conducive to the development of facultative and
anaerobic microorganisms in the deeper layers of the biofilm.
94 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
Figure 4.1 SEM pictures of biofilms.
4.2.2 Processes Involved in Biofilm Development
A number of processes contribute to biofilm development on surfaces exposed to
water flow. The processes involved are the following (Bitton and Marshall, 1980;
Gomez-Suarez et al., 2002; Olson et al., 1991). The sequence of events is described
in Figures 4.2 and 4.3 (Gomez-Suarez et al., 2002; De Kievit, 2009):
4.2.2.1 Surface Conditioning. Surface conditioning is the first step in biofilm
formation. Minutes to hours after exposure of a surface to water flow, a surface-
conditioning layer, made of ions, proteins, glycoproteins, humic-like substances, and
other dissolved or colloidal organic matter, initially adsorb to the surface. This results
in a modified surface that is different from the original one (Schneider and Leis,
BIOFILM DEVELOPMENT IN WDSs 95
(a) (b)
(c) (d)
Conditioning film Transport
Initial adhesion Co-adhesion
(e) (f)Anchoring
Yeasts Bacteria Polymers Conditioning film Surface structures
Growth
Figure 4.2 Sequence of events in biofilm formation Source: Gomez-Suarez et al. (2002).In: Encyclopedia of Environmental Microbiology, Gabriel Bitton, editor-in-chief, Wiley-
Interscience, N.Y.
2002). The conditioning film is a source of nutrients for bacteria, particularly in
oligotrophic environments such as drinking water or groundwater.
4.2.2.2 Transport of Microorganisms to Conditioned Surfaces. Diffu-
sion, Brownian motion, convection, and turbulent eddy transport are involved in
turbulent flow regime. Chemotaxis may also enhance the rate of bacterial adsorption
to surfaces under more quiescent flow.
96 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
1
2 3 4
5
Figure 4.3 Pseudomonas aeruginosa biofilm development. Planktonic cells (stage 1) attach
onto a solid surface (stage 2) and microcolonies are formed (stage 3). Under conditions that
promote bacterial migration (e.g., succinate, glutamate), cells will spread over the substratum,
ultimately developing into a flat, uniformmat (stage 4). Undermotility-limiting conditions (e.g.
glucose), the microcolonies proliferate forming stalk- and mushroom-like structures (stage 4).
At various points throughout biofilm maturation, cells can detach and resume the planktonic
mode of growth (stage 5). Source: De Kievit, T.R. (2009). Environ. Microbiol. 11: 279–288.
4.2.2.3 Adhesion of Microorganisms to Surfaces. According to the ther-
modynamic theory, adhesion of a microorganism to a surface is favored when the
free energy of adhesion is negative (ΔGadh < 0) (Gomez-Suarez et al., 2002). Fur-
thermore, according to the DLVO theory (Derjaguin, Landau, Verwey, and Overbeek
theory), adhesion is a balance between Lifshitz-Van der Waals forces and repulsive
or attractive electrostatic forces due to electrical surface charges on both microbial
and surfaces. Hydrophobic interactions are also involved in microbial adhesion to
surfaces.
4.2.2.4 Cell Anchoring to Surfaces. Following adhesion to a given surface,
microbial cells are anchored to the surface, using extracellular polymeric substances
(EPSs). EPS, made of polysaccharides (e.g., mannans, glucans, uronic acids), pro-
teins, nucleic acids, and lipids help support biofilm structure. EPS display hydrophilic
and hydrophobic properties. They help in the adhesion of microorganisms to surfaces
and their cohesion within biofilms, and play a structural role in biofilms (Bitton
and Marshall, 1980; Characklis and Cooksey, 1983; Costerton and Geesey, 1979;
Flemming and Wingender, 2002; 2010; Starkey et al., 2004; Zhu et al., 2010; Xue
et al., 2013b). EPS also helps protect microorganisms from protozoan predation,
chemical insult, antimicrobial agents, desiccation, and osmotic shock. Moreover,
extracellular polysaccharides chelate heavy metals and reduce their toxicity to
microorganisms (Bitton and Freihoffer, 1978).
Other attachment organelles include flagella, pili (fimbriae), stalks, or holdfasts
(e.g., Caulobacter) (Bitton and Marshall, 1980; Olson et al., 1991).
BIOFILM DEVELOPMENT IN WDSs 97
4.2.2.5 Cell Growth and Biofilm Accumulation. Biofilms essentially con-
sist of microbial aggregates embedded in EPSs attached to a solid surface (Rittmann,
1995b). It was estimated that biofilms harbor about 95% of the microbial communi-
ties in WDSs (Wingender and Flemming, 2004). However, bulk water bacteria may
dominate in portions of the distribution system with no chlorine residual (Srinivasan
et al., 2008).
4.2.3 Factors Involved in Biofilm Accumulation
The study of single-species biofilms has helped in our understanding of the steps
involved in biofilm accumulation which depends on several factors:
4.2.3.1 Concentration of Assimilable Organic Carbon. The presence of
even microgram levels of organic matter in distribution lines allows the growth and
accumulation of biofilm microorganisms (Block et al., 1993; Ollos et al., 2003;
Servais, 1996).
4.2.3.2 Limiting Nutrients. Microbial growth in drinking water can also be
highly regulated by the availability of phosphorus, which can act as a limiting nutrient
for microbial growth in drinking water (Charnock, andKjønnø, 2000;Miettinen et al.,
1997; Keinanen et al., 2002; Sathasivan and Ohgaki, 1999). For example, in most
Finnish drinking waters, microbial growth was well correlated with the concentration
of bioavailable phosphorus (Lehtola et al., 2002; Rubulis and Juhna, 2007). The
presence of bioavailable phosphorus and iron in water distribution pipes also helps
the survival of Escherichia coli (Appenzeller et al., 2005; Juhna et al., 2007). Thus,phosphorous removal by water treatment processes may decrease E. coli survival inthis environment.
4.2.3.3 Trace Elements. Trace elements can stimulate (e.g., Fe, Mn, Zn) or
inhibit (e.g., Cu, Ag) bacterial growth in distribution networks.
4.2.3.4 Disinfectant Concentration. Biofilm thickness and activity are inhib-
ited by disinfection with chlorine. For example, the biofilm thickness (∼103 pg
ATP/cm2) in the presence of a chlorine concentration of <0.05 mg/L was two orders
of magnitude greater than the thickness (∼10 pg ATP/cm2) in the presence of a chlo-
rine level of 0.30 mg/L (Hallam et al., 2001). Attached microorganisms are better
controlled by monochloramine than by free chlorine (see also Chapter 3). In a bench-
scale distribution system, it was found that a free chlorine residual of 0.5 mg/L or
chloramine at 2mg/L reduced biofilmmicroorganisms by several orders ofmagnitude
(Ollos et al., 2003).
4.2.3.5 Type of Pipe Material. Biofilm thickness also depends on the type of
pipe material. Plastic-based materials (polyethylene or PVC) support less attached
biomass than iron (e.g., gray iron), cement-based materials (e.g., asbestos-cement,
cemented cast iron) (Figure 4.4; Niquette et al., 2000), or stainless steel materials.
98 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
Figure 4.4 Effect of pipe material on biofilm accumulation in a drinking water distribution
system. Adapted from Niquette et al. (2000). Water Res. 34: 1952–1956.
Copper pipes showed the lowest biofilm formation potential (Yu et al., 2010). Biofilm
formation, as measured by heterotrophic plate counts and ATP content, was higher
in galvanized steel pipes than in copper pipes, as the release of Cu from the pipes
is toxic to microorganisms (Silhan et al., 2006). Iron corrosion, increased surface
roughness, and porosity in iron pipes contribute to increased attached biomass. Iron,
as a nutrient, is also a factor in increased biomass in the pipes. Pipe material type can
also influence the community composition in the biofilms (Kalmbach et al., 2000).
For example, 74% of the bacteria in PVC pipes were Stenotrophomonas, whereasiron pipes harbored a more diverse microbial populations which consisted mainly of
Nocardia, Acidovorax, Xanthobacter, Pseudomonas, and Stenotrophomonas (Nortonand LeChevallier, 2000). Thus, the use of pipe materials (e.g., stainless steel) that
could reduce biofilm development is desirable (Percival et al., 2000).
4.2.3.6 Flow Velocity and Regimen. An increase in flow rate and a change
from laminar to turbulent flow can enhance the mass transport of nutrients or biocides
into the biofilm, leading to changes in biofilm thickness (see Bachmann and Edyvean
(2006) for more details). Sudden changes in water flow leads to detachment of biofilm
from the pipe surface with subsequent deterioration of water quality due to increase
in suspended bacteria (Choi and Morgenroth, 2003). Hydraulic regime also affects
the bacterial community structure in biofilms. A higher diversity was observed at
highly varied flow in an experimental WDS (Douterelo et al., 2013).
BIOFILM DEVELOPMENT IN WDSs 99
4.2.3.7 Quorum Sensing. A bacterial communication system, which controls
biofilm formation and function (Shrout and Nerenberg, 2012; see more details on this
system in Section 4.2.5.
4.2.3.8 Other Factors. A number of other factors (pH, redox potential, TOC,
water temperature, hardness) control the growth of microorganisms on pipe surfaces
(Olson andNagy, 1984). Temperature affects directly and indirectly the rate of biofilm
formation. A study of biofilm growth in pipes made of different materials showed that
biofilm accumulation was higher at 35◦C than at 15◦C (Silhan et al., 2006). Bacterial
growth generally increases when temperature is above 15◦C although psychtrophic
iron bacteria can grow below this temperature.
4.2.4 Biofilm Ecology
Biofilms allow microorganisms to persist and grow in hostile environments such as
WDSs under low nutrient conditions. In this protected environment, EPSs provide a
diffusional barrier against disinfectants and other deleterious chemicals. EPS increase
the resistance of biofilm and any detached biofilm clusters to chlorine (Xue et al.,
2013b). Using model WDSs, it was found that during the initial stages of biofilm
formation, the 16S RNA sequences in biofilms are similar to those found in the bulk
solution. However, DNA- and RNA-based fingerprints of 20-year-old biofilms show
a higher diversity in biofilm communities than in bulk water (Henne et al., 2012).
The higher diversity in biofilms than in bulk water was also observed in an exper-
imental WDS. Alphaproteobacteria dominated in the bulk water while beta- and
gammaproteobacteria (which include many primary and opportunistic pathogens)
were predominant in biofilms (Douterelo et al., 2013). Microcolonies are formed
following attachment of single cells to the pipe surface. Species diversity increases
thereafter, and biofilms may reach steady state and high ecological diversity, includ-
ing both heterotrophs and autotrophs, only after 2–3 years (Berry et al., 2006;Martiny
et al., 2003). Molecular techniques are now revealing the bacterial diversity in drink-
ing water and biofilms (Kahlisch et al., 2010; Lee et al., 2010; Revetta et al., 2010;
Schmeisser et al., 2003). Molecular probing showed the presence of gram-positive
bacteria as well as alpha-, beta-, and gamma-proteobacteria. Bacteria generally found
in drinking WDSs include Pseudomonas, Acinetobacter, Flavobacterium, Alcali-genes, Aeromonas,Moraxella, Enterobacter,Citrobacter, Sphingomonas,Klebsiella,Burkholderia, Xanthomonas, Methylobacterium, and Bacillus (Berry et al., 2006;
Block et al., 1997). Examination of biofilms from household water meters showed
that the major bacterial genera were Sphingomonas (alpha-proteobacteria), Acidovo-rax, Methylophilus (beta-proteobacteria), and Lysobacter (gamma-proteobacteria).Examination of the Ann Arbor drinking water by 16S rRNA gene sequencing also
showed that most of the bacteria were proteobacteria and belonged to Acidovorax,Variovorax, Sphingopyxis, Ralstonia, and Novosphingobium (Figure 4.5; Lee et al.,
2010). Another study in Ohio, using similar techniques, showed the presence of
proteobacteria, cyanobacteria, actinobacteria, bacteroidetes, and planctomycetes but
57.6% of the sequences were hard to classify (Revetta et al., 2010).
100 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
Bacillus (3%)
E. Coli (3%)
Mesorhizobium
(3%)
Novosphigobium
(6%)
Ralstonia
(6%)
Sphingopyxis
(15%)
Variovorax (42%)
Acidovorax (21%)
Figure 4.5 Bacterial diversity in drinking water. Pie chart shows the relative diversity of
each genus identified by 16S rRNA gene sequencing. Source: Lee et al. (2010). Water Res. 44:
5050–5058.
Bacterial community composition changes following chlorine disinfection as
alpha-, beta-, and gamma-proteobacteria decreased after chlorination (Poitelon et al.,
2010). Community composition is also influenced by the type of disinfectant used
(Hong et al., 2010; Poitelon et al., 2010; Williams et al., 2004; Yu et al., 2010). It is
known that nutrient-depleted environments, such as marine waters and groundwater,
harbor ultramicrocells (UMCs) that pass through 0.2 μm filters (Velimirov, 2001).
Such ultramicrocells were detected in chlorinated drinking water and are part of the
biofilm community. Analysis of 16S rRNA sequences showed that UMC belong to
the Proteobacteria, Firmicules, and Actinobacteria. Some of the UMCs are potential
opportunistic pathogens, such as Stenotrophomonas maltophilia or Microbacteriumsp., that cause infections in immunocompromised patients in hospital setting (Silbaq,
2009).
4.2.5 Gene Exchange and Quorum Sensing in Biofilms
The proximity of microbial cells in biofilms offers opportunities for communication
and exchange of metabolic products and genetic material (e.g., antibiotic resistance
genes) between microorganisms (Cvitkovitch, 2004; Wuertz, 2002; Xi et al., 2009).
The use of modern techniques such as confocal laser scanning microscopy and
fluorescent-labeled (e.g., tagging with green fluorescent protein) plasmids has facili-
tated the study of gene transfer in biofilms and in the environment in general. These
methods have generally shown that gene transfer rates were much higher than those
determined by plating on selective media. In addition to gene transfer by conjugation
and transformation, biofilms can also be a suitable environment for gene transfer via
BIOFILM DEVELOPMENT IN WDSs 101
transduction. An example is the transfer of the Shiga toxin (Stx) gene to E. coli byan Stx-encoding bacteriophage (Solheim et al., 2013).
Much work is presently being done on the molecular mechanisms of biofilm
development. For their survival and interactions in biofilms, bacteria are able to
communicate with cells of the same species or with other prokaryotic and eukaryotic
species. This cell-to-cell communication mechanism, also called “bacterial twitter,”
is quorum sensing which requires a threshold cell density for its triggering. It helps
microorganisms in biofilm development and function and improves their survival by
increasing their access to nutrients and defense against competing microorganisms
(Boyle et al., 2013; de Kievit, 2009; Shrout and Nerenberg, 2012; Williams et al.,
2007). Several quorum sensing bacteria has been found in water and wastewater
treatment systems. These include, among others,Aeromonas,Arcobacter, Legionella,Pseudomonas, Sphingomonas,Vibrio, andNitrobacter (Shrout andNerenberg, 2012).
Quorum sensing diffusible signaling molecules (e.g., N-acyl homoserine lactone),called autoinducers, play an important role in density-dependent gene expression and
biofilm differentiation. Bacteria undergo changes when the signaling molecule has
reached a threshold concentration and when the bacterial population has reached a
certain density. There are three major classes of signaling molecules: acyl homoser-
ine lactones (AHLs), peptides, and LuxS/autoinducer 2. Antibiotics can also func-
tion as signaling molecules at relatively low concentrations (Fajardo and Martınez,
2008). AHL signals are emitted by gram-negative bacteria and their activity was
demonstrated in single-species biofilms (e.g., Pseudomonas aeruginosa) and in nat-urally occurring biofilms (McLean et al., 1997). Peptides are the signaling molecules
in gram-positive bacteria. Autoinducer-2 (AI-2) is another cell-to-cell signaling
molecule that may function in cell communication in some bacteria (Winzer et al.,
2002). Some other functions regulated by the AI-2 signal are biofilm formation
in Bacillus cereus, motility in Helicobacter pylori, virulence factor production in
P. aeruginosa, or susceptibility to antibiotics in Streptococcus anginosus (Pereiraet al., 2013). In addition to biofilm formation, quorum sensing in both gram-negative
and gram-positive bacteria is also known to be involved in the regulation of several
activities, such as virulence, motility, EPS production, sporulation, production of sec-
ondary metabolites such as antibiotics, conjugation, symbiosis, and interspecies com-
petition and other survival strategies (Miller and Bassler, 2001; Withers et al., 2001).
4.2.6 Biofilm Detachment from Surfaces
Biofilm detachment from surfaces releases microorganisms, including pathogens,
which may colonize other surfaces. It affects the occurrence of waterborne diseases
resulting from the transport of opportunistic pathogens to the consumer’s faucet.
Detachment occurs following erosion (detachment of single cells), sloughing (detach-ment of large pieces of biofilm, sometimes extending all the way to the substratum),
scouring following abrasion or scraping and grazing by predators (Percival et al.,
2000; Rittmann, 2004; Rittmann and Laspidou, 2002).
Biological processes also involved in biofilm detachment, include, among other
factors, a decrease in EPS production, rhamnolipids, anti-biofilm poylysaccharides,
102 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
and predation on biofilm microorganisms. Detachment events are genetically regu-
lated (Rendueles et al., 2013; Schooling et al., 2004; Stoodley et al., 2002).
The net accumulation of biofilms on surfaces is described by the following equation
(Rittmann and Laspidou, 2002).
Xf Lf =YJ
b + bdet(4.1)
where;
Xf = biomass density (mg biomass/cm3)
Lf = biofilm thickness (cm)
Y = true yield of biomass (mg biomass/mg substrate)
J = substrate flux (mg substrate/cm2-day)
b = specific decay rate of biofilm microorganisms (d−1)bdet = specific detachment rate (d−1)
Biofilm accumulation increases when the substrate is increased or when detach-
ment is decreased. Under steady state conditions, the average specific growth rate
(μave) is equal to the specific detachment rate (bdet).Slow growing microorganisms (e.g., methanogens) are protected from detachment
because they live in the deeper layers of the biofilm.
There are several factors affecting biofilm detachment (Pedersen, 1990; Rittmann
and Laspidou, 2002). Detachment is:
� increased by tangential flow shear stress which is related to the detachment rate
(at least for smooth surfaces);� increased by axial force on the biofilm, due to abrasion and to pressure changes
caused by turbulent changes and expressed by the Reynolds number Re;� decreased by surface roughness;� affected by physiological parameters such as EPS content (higher detachment
at low EPS) or biofilm density (lower detachment for dense biofilms).
4.2.7 Some Methods Used in Biofilm Study
Several physical, chemical, biochemical, molecular, andmicrobiological methods are
available for biofilms study. Some of these methods, including molecular techniques,
are summarized in Table 4.1 (Broschat et al., 2005; Characklis et al., 1982; Lazarova
and Manem, 1995; Neu and Lawrence, 2002; Percival et al., 2000; Schaule et al.,
2000; Schmidt et al., 2004).
In the cultivation-based method, a low nutrient medium, designated as R2A agar,
is recommended to determine the heterotrophic plate count (HPC) in environmental
samples such as drinking water or groundwater. However, this approach detects only
from 0.1% to 10% of the total bacterial counts in most aquatic environments (Pickup,
1991). Microbial biomass in biofilms can be measured via determination of specific
BIOFILM DEVELOPMENT IN WDSs 103
TABLE 4.1 Some methods used for biofilm study
Type Analytical Method
Microscopy Use of light microcopy, fluorescence microscopy, scanning
confocal laser microscopy, scanning electron microscopy,
environmental scanning electron microscopy, transmission
electron microscopy, atomic force microscopy.
Image analysis Image structure analyzer, time lapse imaging
Direct
measurement of
biofilm quantity
Biofilm thickness (using optical methods, image analysis,
thermal resistance, quartz crystal microbalance)
Biofilm mass (total cell count via staining with Acridine Orange
or DAPI, crystal violet assay)
Indirect
measurement of
biofilm quantity
Extracellular polymeric substances (EPS)
Specific biofilm constituents (polysaccharides, proteins, nucleic
acids)
Total organic carbon (TOC)
Total proteins
Peptidoglycans
Lipid biomarkers
Microbial activity
within biofilms
Viable cell count (plate counts, preferably in low nutrient
medium such as R2A agar, Live/Dead BacLightTM)
Active bacteria: direct viable count (DVC)
DVC-FISH
ATP
Lipopolysaccharides
Substrate removal rate
Dehydrogenase activity (e.g., TTC, INT, or CTC dyes) or
esterases such as carboxyfluorescein diacetate (CFDA).
Oxygen uptake rate (OUR)
DNA
rRNA
mRNA
Microbial
observation and
identification
Immunofluorescence (monoclonal or polyclonal antibodies)
FISH (fluorescent in situ hybridization)mRNA amplified by polymerase chain reaction
Green fluorescent protein (GFP)
Use of commercial fluorochromes such as DAPI, acridine
orange, SYTOX Green, PicoGreen and propidium iodide, or
FUN-1 (for fungi)
Microenvironment Fluor conjugates for determining diffusion and permeability in
biofilms
Use of microelectrodes for determining pH, temperature, O2,
NH4, NO3, NO2, H2S, and CH4 within the biofilm
Other methods Mass spectroscopy
Infrared spectroscopy
X-ray spectroscopy
Source: Adapted from Broschat et al. (2005); Characklis et al. (1982); Lazarova and Manem (1995);
McLean et al. (2004); Mezule et al. (2013); Neu and Lawrence (2002); Percival et al. (2000); Schaule
et al. (2000); Schmidt et al. (2004).
104 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
cell biochemical constituents such as ATP, DNA, RNA, proteins, lipid biomarkers,
bacterial cell wall components, or photosynthetic pigments (Sutton, 2002).
Total cell counts in biofilms are difficult due to the presence of aggregated cells.
This task is made easier by using modern techniques such as scanning confocal
microscopy (SCLM) which gives information about the biofilm structure. The non-
destructive SCLM in conjunction with 16S rRNA-targeted oligonucleotide probes
help to show the heterogeneous structure of biofilms and their complex biodiversity
by giving information on the distribution and activity of specific groups of microor-
ganisms in biofilms (Costerton et al., 1995; Schramm et al., 1996). rRNA-targeted,
fluorescently labeled oligonucleotide probes are useful in identifying biofilmmicroor-
ganisms and community composition (Kalmbach et al., 2000). Other nondestructive
microscopic techniques include epifluorescence microscopy using fluorogenic dyes
(e.g., acridine orange or 6-diamidino-2-phenylindol known as DAPI), transmission
electron microscopy (TEM), scanning electron microscopy (SEM), and environ-
mental SEM (ESEM) which allows the observation of the specimen in its own
hydrated state.
Microelectrode probes give information on the physicochemical properties (pH,
temperature, O2, NH4, NO3, NO2, H2S, CH4) of the biofilm microenvironment
(Burlage, 1997). These microelectrodes have helped shed light on the distribution
of nitrifiers and sulfate reducers along the depth of biofilms. Microelectrodes for
biofilm study will be replaced in the future with fiber-optic microsensors (Beyenal
et al., 2000).
Microbial activity within biofilms can also be assessed by measuring enzyme
activity (e.g., esterase or dehydrogenase activity), reduction of tetrazolium salts such
as cyanoditolyl tetrazolium chloride (CTC), 2-p-(iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium chloride (INT), triphenyl tetrazolium chloride (TTC), or resorufin by
biofilmmicroorganisms. Fluorescein diacetate (FDA) or carboxyfluorescein diacetate
(CFDA) are used to measure esterase activity and the active fluorescent cells are
counted under a fluorescent microscope. Cell viability can also be determined by the
LIVE/DEAD BacLight kit, or special dyes which measure membrane integrity (e.g.,
SYBR Green propidium iodide). The fluorescence intensity can be determined via
flow cytometry (Berney et al., 2007, 2008; Cerca et al., 2011).
Several techniques have been proposed for gaining information about biofilm
formation and thickness. Some of the methods are (Broschat et al., 2005; Milferstedt
et al., 2006; Nivens et al., 1995; Reipa et al., 2006):
� Quartz crystal microbalance (QCM). This nondestructive technique measures
biomass changes on a quartz crystal. Both the quartz oscillator frequency (f)and resistance (R) are monitored during biofilm formation and the data are
expressed as ΔR/Δf which reflects changes in the viscoelastic properties of thebiofilms. QCM was used in the long-term monitoring of biofilm formation by
Pseudomonas cepacia and P. aeruginosa.� Optical methods: Early on, biofilm thickness was estimated by focusing a light
microscope on the top and bottom layers of a biofilm. A more recent approach
GROWH OF PATHOGENS AND OTHER MICROORGANISMS IN WDSs 105
consists of exposing the biofilm to a desktop scanner and then following with
image analysis. The gray level of the images is well correlated with the biofilm
biomass. A reflectance assay was considered to determine biofilm formation of
14 Enterococcus isolates on opaque and nonopaque surfaces� Crystal violet assay: This method consists of staining dry biofilms with 0.1%crystal violet, washing with buffer to remove excess dye, releasing the dye from
the biofilm with 95% ethanol and measuring the absorbance at 595 nm.
4.3 GROWH OF PATHOGENS AND OTHER
MICROORGANISMS IN WDSs
Pathogen accumulation in biofilms of WDSs is an important public health issue as
it may contribute to the spread of waterborne diseases. Pathogens enter the WDS
through insufficient water treatment or through leaks. They grow in WDSs and
colonize pipe inner walls, connections, tubercles, and dead ends. However, biofilm
growth over surfaces is not continuous as noncolonized spots are observed. Bacterial
accumulation preferably occurs in the colonized area and increases with biofilm age
(Paris et al., 2009).
4.3.1 Earlier Studies on the Microbiology of Distribution Systems
The presence of a wide range of microorganisms (eubacteria, filamentous bacteria,
actinomycetes, diatoms) was demonstrated in distribution pipes by SEM. Some of
the bacteria were seen attached to the surfaces by means of extracellular fribrillar
materials (Ridgway andOlson, 1981; Ridgway et al., 1981). Turbercles provide a high
surface area for microbial growth and protect microorganisms from the lethal action
of disinfectants. Turberculated cast iron pipe sections from the WDS in Columbus,
Ohio, metropolitan area harbored high numbers of aerobic and anaerobic bacteria
(e.g., sulfate reducing bacteria). Some samples contained up to 3.1 × 107 bacteria per
gram of tubercle material (Tuovinen and Hsu, 1982). Several investigators have also
reported the proliferation of iron and manganese bacteria that grow attached to pipe
surfaces and, subsequently, cause a deterioration of water quality (e.g., pipe clogging
and color problems). Attached iron bacteria such as Gallionella have stalks that arepartially coveredwith iron hydroxide. This was confirmed byX-ray energy-dispersive
microanalysis (Ridgway et al., 1981).
4.3.2 Opportunistic Bacterial Pathogens
As mentioned in Chapter 1, this group includes genera such as Pseudomonas,Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acine-tobacter, Proteus, Providencia, L. pneumophila, S. maltophilia, and nontubercular
mycobacteria (NTM) (Sobsey and Olson, 1983; van der Wielen and van der Kooij,
106 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
2013). They are of health concern to the young, the elderly, and immunocompromised
consumers. The routine occurrence of these opportunistic pathogens in distribution
systems have been documented in the literature (Keevil, 2002). For example, most
Aeromonas isolates from 13 Swedish drinking WDSs produced virulence factors
such as cytotoxins, suggesting potential public health problems (Kuhn et al., 1997).
Sphingomonas spp. can also be found in water distribution biofilms. They are oftenoverlooked due to their slower growth than other members of the HPC (Koskinen
et al., 2000). They have been isolated from activated carbon and sand filters, and are
potential reservoirs of antibiotic-resistant bacteria in drinking water (Vaz-Moreira
et al., 2011). Biofilms can also be a potential reservoir for H. pylori which can sur-vive in the viable but nonculturable (VBNC) state (Linke et al., 2010; Percival and
Thomas, 2009). The VBNC state is a bacterial response to stress (e.g., nutritional
stress, exposure to disinfectants and other toxicants such as heavy metals). A wide
range of bacteria are now known to enter in the VBNC state when exposed to envi-
ronmental stress (Oliver, 2005). Biofilmsmay also harbor antibiotic-resistant bacteria
(Schwartz et al., 2003; see also Chapter 1).
4.3.3 Nontubercular Mycobacteria (NTM)
The genusMycobacterium includes pathogenic species such asM. tuberculosum and
M. leprae, and NTM which are found in soil and aquatic environments and are con-
sidered as opportunistic pathogens (Falkinham, 2002; Falkinham et al., 2001). NTM
were detected around the world in 21% to more than 80% of samples from WDSs
(see review by Vaerewijck et al., 2005). A more recent survey in two chloraminated
WDSs in Florida and Virginia showed that the percent occurrence ofMycobacteriumspp. was 93.7% and 94.4%, respectively. The percent occurrence of M. avium was
10% and 8.9%, respectively (Wang et al., 2012b). In Germany and France, mycobac-
terial species were found in 90% of biofilm samples taken from water treatment
plants (Schulze-Robbecke et al., 1992). A survey of seven water treatment plants in
the Netherlands showed that NTM were detected in all distributed drinking water
samples and their levels were much higher in distributed water than in treated water
(van der Wielen and van der Kooij, 2013). Approximately one-third of the 100
known mycobacterial species have been detected in WDSs. In the Netherlands, sev-
eral mycobacterial species (M. peregrinum, M. salmoniphilum, M. llatzerense, andM. septicum) were identified in distributed water and in shower water (van Ingen
et al., 2010). The primary Mycobacterium species detected in biofilms of WDSs
in China (Guangzhou and Beijing) were M. arupense and M. gordonae (Liu et al.,2012a). Mycobacterium avium subsp. paratuberculosis (MAP), a possible causative
agent of Crohn’s autoimmune disease in humans, was detected, via PCR, in finished
drinking water (Aboagye and Rowe, 2011; Beumer et al., 2010). This opportunistic
pathogen persists for several weeks in biofilms and its survival is much higher than
that of E. coli (Lehtola et al., 2007; Torvinen et al., 2007). The growth of M. aviumin WDSs depends on the level of available organic carbon (Falkinham et al., 2001;
LeChevallier, 2004).
GROWH OF PATHOGENS AND OTHER MICROORGANISMS IN WDSs 107
4.3.4 Legionella in Hot-Water Tanks and Distribution Systems
Due to airborne transmission through showering, much work has been carried out on
the survival and growth of Legionellae in potable WDSs and plumbing in hospitals
and homes (Colbourne et al., 1988; Felfoldi et al., 2010; Muraca et al., 1988). A
recent survey showed that the percent occurrence of Legionella spp. was 67.5% in
a Florida WDS and 30% in a Virginia system (Wang et al., 2012b). Furthermore,
legionellae appear to be more resistant to chlorine than E. coli (States et al., 1989),and small numbers may survive in distribution systems that have been judged to be
microbiologically safe. Under high shear turbulent-flow conditions, survival of L.pneumophila and Mycobacterium avium is much higher than that of E. coli whichis traditionally used as an indicator (Lehtola et al., 2007). Legionellae may also
grow to detectable levels inside hot-water tanks in hospitals and homes and thus
pause a health threat (Stout et al., 1985; Witherell et al., 1988). The Legionellaisolates responsible for nosocomial Legionnaires’ disease corresponded to isolates
from potable water (Garcia-Nunez et al., 2008). Legionella survives well at 50◦C
and may even grow and multiply in tap water at 32–42◦C (Dennis et al., 1984;
Yee and Wadowsky, 1982). The enhanced survival and growth in these systems
have also been linked to stagnation (Ciesielski et al., 1984), stimulation by rubber
fittings in the plumbing system (Colbourne et al., 1988), and trace concentrations
of metals such as Fe, Zn, and K (States et al., 1985, 1989). It was found that
sediments found in WDSs and tanks (i.e., scale and organic particulates) and the
natural microflora significantly improved the survival of Legionella pneumophila.Sediments indirectly stimulate Legionella growth by promoting the growth of com-mensalistic microorganisms (Stout et al., 1985; Wadowsky and Yee, 1985). For
example, Mycobacterium chelonae, an opportunistic bacterial pathogen, can have apositive effect on the cultivability of L. pneumophila and H. pylori in biofilms (Giaoet al., 2011).
Protozoa, mostly amoebas and ciliates, play a role in the survival of Legionellain water distribution pipes. Biofilms and the presence of protozoa (e.g., Acan-thamoeba spp.) play an essential role in the survival, proliferation, and pathogenicityofLegionella in drinkingwater (Corsaro et al., 2010; Lau andAshbolt, 2009). Biofilmsgrowing in in-premise plumbing harbor Legionella cells as well as protozoan hostswhich help in the propagation of this pathogen. The biofilm-associated Legionella isdetached and aerosolized during showering and can be subsequently inhaled.
Several methods have been considered for controlling Legionella in water: super-chlorination (chlorine concentration of 2–6 mg/L), chloramination, maintaining the
hot-water tanks at temperatures above shock treatment at 70◦C for 30minutes, ultravi-
olet irradiation (Antopol and Ellner, 1979; Knudson, 1985; Kool et al., 2000; Muraca
et al., 1987; Stout et al., 1986), biocides (Fliermans and Harvey, 1984; Grace et al.,
1981; Soracco and Pope, 1983), and alkaline treatment (States et al., 1987). There
are less Legionella nosocomial infection outbreaks in hospitals that use monochlo-ramine than those using free chlorine (Flannery et al., 2006; Heffelfinger et al., 2003;
Kool et al., 1999). Legionella can, however, become heat-resistant after repeated heattreatments (Allegra et al., 2011). The control of legionellae in potable water systems
108 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
may also be achieved via the control of protozoa which may harbor these pathogens
(States et al., 1990).
4.3.5 Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes
Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) to sev-
eral antibiotics (amoxilline, chloramphenicol, ciprofloxacin, gentamicin, rifampin,
sulfisoxasole, tetracycline) were found in several finished and tap water samples.
The ARB levels were often higher in tap water than in finished water, indicating
that WDSs can serve as a reservoir for ARB and ARG (Xi et al., 2009). Antibiotic
resistance genes have been detected in biofilms from drinkingWDSs. Enterobacterial
ampC β-lactam-resistance and enterococcal vanA vancomycin-resistance genes were
detected in drinking water biofilms although no enterobacteriaceae or enterococci
were detected in those samples. However, the Staphylococcal mecA methicillin-
resistance gene was not found in drinking water biofilms (Schwartz et al., 2003).
Several studies have shown that chlorination of water and wastewater induces
selection for ARB (Murray et al., 1984; Shrivastava et al., 2004). The mechanism of
this selection is not well known.
However, at the present time, we do not know if increased resistance to antibi-
otic present a significant risk to consumers, especially the young, the elderly, and
immunocompromised people.
Table 4.2 lists some human pathogens and opportunistic pathogens detected in
biofilms of domestic plumbing systems (Eboigbodin et al., 2008).
4.3.6 Protozoan Parasites
Biofilms can also serve as reservoirs for protozoan parasites cysts and oocysts. The
trapping and concentration of C. parvum oocysts and their survival in biofilms was
demonstrated under laboratory conditions (Rodgers and Keevil, 1995). Free-living
TABLE 4.2 Some human pathogens detected in biofilms from domestic plumbingsystems
Pathogen Comments
Legionella pneumophila Causes Legionnaires’ disease
Legionella bozemanii Causes human pneumonia
Helicobacter pylori Causes gastric diseases including cancer
Pseudomonas sp. Some species (e.g., P. aeruginosa, P. maltophilia) arehuman pathogens
Sphingomonas sp. Some species cause nosocomial infections in humans
Micrococcus kristinae Found in catheter-associated bacteremia
Corynebacterium sp. Part of human skin flora. Some species are pathogenic
Acanthamoeba keratitis Can cause infection in the cornea
Source: Adapted from Eboigbodin et al. (2008). Amer. Water Works Assoc. J. 100 (10): 131–137.
SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS IN DRINKING WATER 109
pathogenic protozoa, such as Naegleria fowleri, may also find refuge in biofilms,
particularly in stagnant areas of the distribution network. There is experimental
evidence thatN. fowleri can colonize and persist for months in pipe biofilms, althoughnone was detected in two full-scale distribution networks (Biyela et al., 2012).
The cysts and oocysts can subsequently be resuspended in WDSs by biofilm
sloughing following an increase in water flow (Helmi et al., 2008; Puzon et al., 2009;
Searcy et al., 2006).
4.3.7 Enteric Viruses
Enteric viruses are occasionally detected in treated drinking water (Bitton et al.,
1986). Laboratory studies have shown that, following their entry into the WDS,
viruses may adsorb to biofilms where they may survive for relatively long peri-
ods. A similar phenomenon was observed in wastewater systems where viruses
(noroviruses, enteroviruses, FRNA phages) were found to attach and persist in
biofilms for longer periods than in wastewater (Skraber et al., 2009). However, only
one of three field studies of virus monitoring in biofilms showed the presence of infec-
tious enteroviruses (Vanden Bossche, 1994). Similarly to other pathogens, pathogenic
viruses may contaminate drinking water through sloughing of biofilm pieces.
4.4 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
IN DRINKING WATER TREATMENT AND DISTRIBUTION
The development of biofilms on surfaces can be beneficial or detrimental to processes
in water and wastewater treatment plants. Trickling filters and rotating biological
contactors are examples of processes that rely on microbial activity in biofilms to
treat wastewater.
4.4.1 Advantages
Some advantages offered by biofilms are (Rittmann, 1995b; Shrout and Nerenberg,
2012)
� The fact that anytime a solid surface is exposed to water is followed by biofilm
formation demonstrates well the benefits of biofilms to microorganisms.� Biofilms act as biocatalysts in natural systems for the self-purification of surface
waters and groundwater, and in engineered fixed-film processes (e.g., trickling
filters, rotating biological contactors, biofilters in water purification, riverbank
filtration, membrane-based biofilm reactors) for the treatment of water and
wastewater.� They are desirable whenever microorganisms must be retained (i.e., need for a
high mean cell residence time) in a system with a short hydraulic retention time,
110 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
as is the case for biological treatment of drinking water which involves the pro-
cessing of large volumes of water with very low concentrations of biodegradable
compounds.� Many of the processes used in drinking water treatment allow biofilm formation
and include, among others, granular activated carbon, slow and rapid sand filters,
and biological water treatment.
4.4.2 Disadvantages
However, biofilms can cause problems in water treatment and distribution systems
(Rittmann, 1995b, 2004; Smith, 2002; van der Wende and Characklis, 1990):
� Because of mass-transport resistance, bacteria are exposed to lower concentra-
tions of substrates than in the bulk liquid.� Biofilm accumulation increases fluid frictional resistance in distribution
pipelines (Figure 4.6; Bryers and Characklis, 1981), leading to an increase
in pressure drop and to reduced water flow if the pressure drop is held con-
stant. The microbial community structure of the biofilm also affects frictional
resistance. A predominantly filamentous biofilm appears to increase frictional
resistance (Trulear and Characklis, 1982).� Biofouling decreases flow rates in membrane-based water treatment processes.
The biofouling is due to the overproduction of EPSs mainly composed of
polysaccharides.
GROWTHINDUCTION
TIME
PLATEAU
Biofilm mass
or thickness
Frictional
resistance
Heat transter
resistance
Figure 4.6 Increase in heat transfer resistance and frictional resistance resulting from biofilm
growth. Source: Bryers and Characklis (1981). Water Res. 15: 483–491.
SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS IN DRINKING WATER 111
� Anaerobic conditions lead to the production of H2S, a toxic gas characterized
by a rotten-egg odor.� Biofilms have an impact on public health and are the cause of persistent infec-
tions (Costerton, 1999). They are conducive to the accumulation of pathogens
and parasites in distribution pipes biofilms which are habitats for opportunistic
pathogens such as Legionella andMycobacterium and lead to infections related
to implants and dental plaques. Biofilms are involved in 80% of chronic inflam-
matory and infectious diseases due to pathogenic bacteria (Sauer et al., 2007).
Biofilms developing on implanted medical devices harbors microbial pathogens
such as Candida albicans, Staphylococcus aureus, P. aeruginosa, Kelbsiellapneumoniae, and Enterococcus spp., and E. coli. Most of the circulatory and
urinary tract infections are due to biofouled implanted devices. These infec-
tions increase patient morbidity and mortality and the cost of medical care.
Biofilms associated with medical implants are often resistant to antibacterial
and antifungal drugs (Anwar and Costerton, 1992; Donlan, 2002; Thomas et al.,
2004).� Corrosion problems: Biocorrosion of pipes is associated with biofilm growth.
Biofilm accumulation is associated with corrosion of iron pipes where Fe2∗
serves as an electron donor. Complaints about red and black waters, due to the
activity of iron- and manganese-oxidizing bacteria (e.g., Gallionella, Hyphomi-crobium) (Rittmann, 2004).
� Resistance of biofilm microorganisms to biocides: Biofilm microorganisms can
be up to 1500 times more resistant to biocides and antibiotics than freely sus-
pended bacteria (Sauer et al., 2007). Increase in resistance to chlorine dis-
infection contributes to the regrowth of indicator and pathogenic bacteria in
distribution systems. It was suggested that this increased resistance may be due
to diffusional resistance of the biofilmmatrix as measured with a chlorinemicro-
electrode (de Beer et al., 1994), the protective effect by extracellular polymeric
materials, the presence of efflux pumps that export biocides, antibiotics, and
other toxic substances to the bacterial surrounding medium (Marquez, 2005),
selection of biofilmmicroorganismswith enhanced resistance to the disinfectant,
attachment of bacteria to biological (e.g., macroinvertebrates and algal surfaces)
and nonbiological surfaces (particles, activated carbon) or cell surface hydropho-
bicity (Steed and Falkinham, 2006). Furthermore, multispecies biofilms appear
to be more resistant to disinfection than single-species ones (Simoes et al.,
2010). As shown for activated carbon, biofilm bacteria appear to be more resis-
tant to residual chlorine and monochloramine than suspended bacteria (Berry
et al., 2009; LeChevallier et al., 1988). The protective effect of biofilms toward
pathogens exposed to chlorine was also demonstrated forMycobacterium aviumand Mycobacterium intracellulare (Steed and Falkinham, 2006). Particulates,
including particulate organic matter, protect pathogenic microorganisms from
disinfectant action (Hoff, 1978). It was found that Enterobacter cloacae, in thepresence of particulates fromWDSs, is protected from chlorine action as a result
of its attachment to the particles (Herson et al., 1987). Chloramines appear to
112 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
be more efficient in biofilm control than free chlorine (HOCl or OCl−) (seeChapter 3). This may be due to the lower affinity of chloramines for bacte-
rial polysaccharides (LeChevallier et al., 1990; van der Wende and Characklis,
1990). It was proposed that, for biofilm control, free chlorine should be used
as a primary disinfectant but the residual should be converted to chloramine
(LeChevallier et al., 1990). The increased resistance of biofilm microorganisms
to chlorine applies to other antibacterial agents such as antibiotics. The free
chlorine residual decreases as the water flows through the distribution system.
In a laboratory study, silver (100 μg/L) used as a biocide did not have any signif-icant effect on preventing biofilm formation on PVC and stainless steel surfaces
(Sylvestry-Rodriguez et al., 2008).
4.5 BIOFILM CONTROL AND PREVENTION
Biofilms can be controlled or removed by mechanical, chemical, or enzymatic means
(Brisou, 1995). The drinking water industry has a few options for controlling biofilm
growth in WDSs (Camper et al., 2003; Rittmann, 2004):
� Mechanical cleaning: It is combinedwith the use of biocideswhen the biofouling
is severe.� Maintain a proper level (1 mg/L as Cl2) of chlorine residual throughout the
WDS to reduce biofilm accumulation. The chlorine level cannot exceed 4 mg/L
as Cl2. Maintaining a chlorine residual is, however, difficult to achieve in large
systems and cannot prevent biofilm growth in the presence of high levels of
electron donors.� Obtain a biostable water by removing or drastically reducing the level of organic
and inorganic electron donors in treated water. This can be accomplished by
using membrane filtration, enhanced coagulation, or biological filtration (see
Chapter 6).� Consider the effect of the pipe material: Replace deteriorating iron pipes with
other materials (e.g., PVC or CPVC pipes). However, one should be concerned
about the leaching and accumulation of the monomer, vinyl chloride, a proven
human carcinogen (Walter et al., 2011).� Potential “jamming” of quorum sensing in biofilms (Barraud et al., 2009;
Hentzer et al., 2004; Shrout and Nerenberg, 2012): Attempts to interrupt quorum
sensing in biofilms are potentially a useful strategy in the control of infectious
diseases. As a result of the emergence of antibiotic resistance in bacteria, there
is presently a quest for quorum sensing inhibitors to attenuate and control bacte-
rial infections (Hentzer et al., 2003). Laboratory studies have shown that several
chemicals can control biofilms by interrupting quorum sensing. Some examples
are 2,4-dinitrophenol, a furanone produced by red algae, nitric oxide to dis-
perse single- or multi-species biofilms, or protoanemonin, a natural compound
which inhibits quorum sensing in P. aeruginosa. The application of this research
BIOFILM CONTROL AND PREVENTION 113
0
10
20
30
40
50
60
70
80
90
100
PAβNNMPT
Bio
film
fo
rma
tio
n r
ela
tive
to
co
ntr
ol w
ith
ou
t E
PIs
Figure 4.7 Effect of efflux pump inhibitors (T, NMP and PAβN) on biofilm formation in
Staphylococcus aureus. T = Thioridazine; NMP = 1-naphthylmethyl piperazine; PaβN =Phe-Arg-β Naphthylamide. Adapted from Kvist et al. (2008). Appl. Environ. Microbiol. 74:
7376–7382.
to WDSs under field conditions awaits further investigation. Quorum sensing
interruption by chemicals could be used in combination with other control mea-
sures to improve biofilm control. Somemicroorganisms make endogenous nitric
oxide (NO)which is generated byNO-synthase under the control of the nos gene.The presence of NO helped decrease the ability of P. putida to form biofilms
(Bobadilla Fazzini et al., 2013; Liu et al., 2012b; Njoroge and Sperandio, 2009).
There are also quorum enzymes or bacteria that release enzymes that inhibit
signaling in biofilms, leading to control of biofouling in membrane bioreactors
(Jiang et al., 2013; Kim et al., 2013b). As an example, a recombinant E. colithat produces N-acyl homoserine lactonase, inhibited biofouling in a membranebioreactor (Oh et al., 2012; Yeon et al., 2009a, 2009b). The feasibility of this
approach for controlling biofilms is yet to be demonstrated.� As regard the formation of biofilms on medical devices, great efforts are
being made to prevent their formation and to develop drugs to treat existing
biofilms. One original control method is to block quorum-sensing pathways in
order to prevent the formation of biofilms on medical devices. Bacteria also
have several chromosome-encoded efflux pumps that help export antibiotics,
antiseptics, heavy metals, solvents, detergents, and other biocides to the sur-
rounding medium (Martinez et al., 2009). These efflux pumps would lead to
increased resistance of biofilms to toxic chemicals, including disinfectants. The
114 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS
addition of efflux pumps inhibitors (e.g., thioridazine, phe-arg β-naphthylamide,1-naphthylmethyl piperazine) helped reduce biofilm formation by Staphylococ-cus aureus (Figure 4.7; Kvist et al., 2008). A wide range of chemicals are being
screened by biotechnological companies to find a way to control biofilms. Some
of these chemicals are the halogenated furanones produced by marine algae
(Schachter, 2003).� Antibiofilm polysaccharides: Some bacteria are able to produce antibiofilm
polysaccharides which can inhibit biofilm formation or destabilize preformed
biofilms. They potentially have useful applications in industrial and medical
fields and can help reduce the incidence of infections caused by biofilms on
medical devices (Rendueles et al., 2013).� Other biofilm control approaches are surface modification or application of an
electric current (Chiang et al., 2009; Wellman et al., 1996).
WEB RESOURCES
http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/
(biofilm online manual, Amer. Soc. Microbiology)
http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/primer.html
(biofilm primer)
http://www.erc.montana.edu/
(Center for Biofilm Engineering, Montana State University, Bozeman)
http://www.nesc.wvu.edu/ndwc/articles/OT/SP01/History_Distribution.html
(history of drinking water distribution)
http://www.nottingham.ac.uk/quorum/
(general information about quorum sensing)
http://www.mrc-lmb.cam.ac.uk/genomes/awuster/wecb/
(gives many references on quorum sensing)
http://www.epa.gov/safewater/dwh/index.html
(Drinking water: EPA consumer information)
http://www.epa.gov/safewater/sdwa/index.html
(SDWA: Safe Drinking Water Act)
http://www.epa.gov/safewater/standards.html
(General information on drinking water)
http://www.cdc.gov/safewater/
(Safe Water System, Center for Disease Control and Prevention)
FURTHER READING 115
FURTHER READING
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of its causes, consequences and control in drinking water distribution systems. Biofilms2:197–227.
Berry, D., C.Xi, andL. Raskin. 2006.Microbial ecology of drinkingwater distribution systems.
Curr. Opin. Biotechnol. 17:297–302.Costerton, J.W., Z. Lewandowski, D.E. Caldwell, D.R. Korber, and H.M. Lappin-Scott. 1995.
Microbial biofilms. Annu. Rev. Microbiol. 49:711–745.Donlan, R.M. 2002. Biofilms: microbial life on surfaces. Emerging Infect. Dis. 8:880–890.Eboigbodin, K.E., A. Seth, and C.A. Biggs. 2008. A review of biofilms in domestic plumbing.
Am Water Works Assoc. J. 100(10):131–137.Flemming, H.-C. and J. Wingender. 2010. The biofilmmatrix.Nat. Rev. Microbiol. 8:623–633.Ghannoum, M. and G.A. O’Toole (Eds). 2004. Microbial Biofilms. ASM Press, Washington,
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Keevil, C.W. 2002. Pathogens in environmental biofilms. In Encyclopedia of EnvironmentalMicrobiology, G. Bitton (editor-in-chief). Wiley-Interscience, New York, pp. 2339–2356.
LeChevallier, M.W., M.-C. Besner, M. Friedman, and V.L. Speight. 2011. Microbiological
quality control in distribution systems. In Water Quality and Treatment: A Handbook onDrinking Water, 6th edition, J.K. Edzwald (Ed.). AWWA, Denver, CO, pp. 21.1–21.84.
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Peirera, C.S., J.A. Thompson, and K.B. Xavier. 2013. AI-2-mediated signaling in bacteria.
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Drinking Water. CRC Press, Boca Raton, FL, 229 pp.
Rendueles, O., J.B. Kaplan, and J.-M. Ghigo. 2013. Antibiofilm polysaccharides. Environ.Microbiol. 15:334–346.
Rittmann, B.E. 2004. Biofilms in the water industry. InMicrobial Biofilms, M. Ghannoum and
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